Tissue-adhesive porous haemostatic product

ABSTRACT

A process of preparing an adhesive haemostatic product is provided. The process comprises: (a) coating a porous solid substrate with a coating liquid that comprises an electrophilically activated polyoxazoline (EL-POX) and a solvent to produce a coated substrate; and (b) removing the solvent from the coated substrate. The EL-POX comprises at least 2 reactive electrophilic groups. The process enables the application of an EL-POX coating that leaves the pore structure of the substrate largely intact so that the ability of the porous substrate to absorb body fluids, such as blood, remains essentially unaffected. The EL-POX coated haemostatic product obtained by the present process has excellent adhesive properties due to the presence of electrophilic reactive groups that are capable of reacting with e.g. amine groups that are naturally present in tissue, under the formation of covalent bonds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Division of U.S. application Ser. No. 15/516,467,filed Apr. 3, 2017, which is the National Phase of International PatentApplication No. PCT/NL2015/050696, filed Oct. 5, 2015, published on Apr.14, 2016 as WO 2016/056901 A1, which claims priority to European PatentApplication No. 14187781.1, filed Oct. 6, 2014. The contents of theseapplications are herein incorporated by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a tissue-adhesive haemostatic productcomprising a porous solid substrate and a coating that contains anelectrophilically activated polyoxazoline (EL-POX). This new haemostaticproduct exhibits excellent biodegradability, adhesiveness andhaemostatic properties. Examples of haemostatic products encompassed bythe present invention include haemostatic meshes, haemostatic foams andhaemostatic powders.

Also provided is a process for preparing a tissue-adhesive haemostaticproduct which involves coating a porous solid substrate with anelectrophilically activated polyoxazoline (EL-POX).

BACKGROUND OF THE INVENTION

Wound dressings form an important segment of the global wound caremarket. These products are widely used in the treatment of injuries suchas wounds, hemorrhages, damaged tissues, and bleeding tissues. The idealdressing should prevent excessive bleeding and promote rapid healing ata reasonable cost with minimal inconvenience to the patient.

The haemostatic properties of a wound dressing are determined by thetexture and the porosity of the material. As regards porosity, the poresof the dressing are usually so tiny that they are not visible to thehuman eye upon casual inspection. They are, however, of sufficient sizenot only to permit ample transpiration of skin moisture and woundvapors, but also to permit absorption of blood so that the dressingbecomes firmly anchored to the tissue once the blood has coagulated.

Wound dressings should be able to maintain a moist environment aroundthe wound, effective oxygen circulation to aid regeneration of cells andtissue, and a low bacterial load. Wound dressings that are employedduring surgery and that remain in the body should be biodegradable andcompletely resorbable.

Conventional tissue-adhesive wound dressings include fibrin sealants,cyanoacrylate based sealants, and other synthetic sealants andpolymerizable monomers. These tissue-adhesives are only suitable forspecific applications because of several drawbacks, including release oftoxic degradation products, high cost, need for refrigerated storage,slow curing, limited mechanical strength and risk of infection.Therefore, hydrogel tissue adhesives have been developed on the basis ofreactive polyethylene glycol (PEG) precursors. However, these hydrogeltissue adhesives swell or dissolve away too quickly, or lack sufficientcohesion, thereby decreasing their effectiveness as surgical adhesive.Moreover, the properties of such PEG-based material cannot be easilycontrolled.

Haemostatic powders are another example of a haemostatic product that iswidely used. Examples of commercially available haemostatic powers, alsoknown as styptic powders, include an adsorbable haemostatic gelatinpowder (Spongostan® powder) and a calcium-loaded form of zeolite alsoknown as QuikClot®. These haemostatic powders can be used to stop severebleeding.

U.S. Pat. No. 5,614,587 describes collagen-based compositions useful inthe attachment of tissues, or the attachment of tissues to syntheticimplant materials. The compositions comprise fibrillar collagen, a fiberdisassembly agent, and a multifunctionally activated synthetichydrophilic polymer such as polyethylene glycol, wherein the collagenand synthetic polymer covalently bind to form a collagen-syntheticpolymer conjugate.

WO 2004/028404 describes a tissue sealant composed of a syntheticcollagen or synthetic gelatin and a electrophilic cross-linking agentwhich are provided in a dry state. In this international publication thecrosslinker comprises an electrophilically activated (EA) poly (ethyleneglycol) (PEG) or an EA PEG derivative such as PEG-succinimidyl ester, inparticular PEG-succinimidyl propionate, PEG-succinimidyl butanoate, orPEG-succinimidyl glutarate. Upon wetting of this composition at anappropriate pH a reaction between the 2 components takes place and a gelwith sealing properties is formed.

US 2011/0251574 describes a haemostatic porous composite spongecomprising a matrix of a biomaterial and a hydrophilic polymericcomponent comprising reactive groups wherein said polymeric component iscoated onto a surface of said matrix of a biomaterial, or said matrix isimpregnated with said polymeric material, or both. In a preferredembodiment the polymer is a polyalkylene oxide polymer, moreparticularly a multi-electrophilic polyethylene glycol (PEG). The matrixmaterial can be selected from collagen, gelatin, fibrin, apolysaccharide (such as chitosan), a synthetic biodegradable biomaterial(such as polylactic acid or polyglycolic acid), and derivatives thereof.

US 2010/069579 a terminally activated polyoxazoline (POZ) compound, saidPOZ compound comprising a POZ polymer having a single active functionalgroup on a terminal end thereof, said functional group capable ofreacting with a group on a target molecule to create a targetmolecule-POZ conjugate wherein all the linkages between the targetmolecule and the POZ compound are hydrolytically stable linkages.

WO 2012/057628 describes crosslinked polyoxazoline polymers havingtissue-adhesive properties due to the presence of electrophilic groupsthat are capable of reacting with nucleophile-containing chemicalentities present in natural tissue.

It is an object of the present invention to provide a tissue-adhesivehaemostatic product with improved properties.

SUMMARY OF THE INVENTION

The inventors have discovered that a tissue-adhesive haemostatic producthaving improved properties can be produced by a process that comprisesto following steps:

-   -   providing a porous solid substrate;    -   coating the substrate with a coating liquid that comprises an        electrophilically activated polyoxazoline (EL-POX) and a solvent        to produce a coated substrate, said EL-POX containing at least 2        reactive electrophilic groups;    -   removing the solvent from the coated substrate.

The inventors have surprisingly found that EL-POX can be applied onto aporous solid substrate by means of the present method without adverselyaffecting the porous structure of the substrate. The present processenables the application of an EL-POX coating that leaves the porestructure of the substrate largely intact so that the ability of theporous substrate to absorb body fluids, such as blood, remainsessentially unaffected. The EL-POX coated haemostatic product obtainedby the present process has excellent adhesive properties due to thepresence of electrophilic reactive groups that are capable of reactingwith e.g. amine groups that are naturally present in tissue, under theformation of covalent bonds.

The present invention also provides a tissue-adhesive haemostaticproduct selected from a coated mesh, a coated foam and a coated powder,said haemostatic product comprising:

-   -   a porous solid substrate having a porosity of at least 5 vol. %        and comprising an outer surface that comprises a nucleophilic        polymer containing reactive nucleophilic groups;    -   an adhesive coating that covers at least a part of the solid        substrate, said coating comprising an electrophilically        activated polyoxazoline (EL-POX), said EL-POX containing on        average at least 1 reactive electrophilic group.

The EL-POX polymer contained in the coating of the haemostatic productoffers the advantage that it can carry a high number of electrophilicreactive groups due to the fact that the EL-POX polymer can contain alarge number of pendant groups that each can carry one or more of suchreactive groups. Thus, the adhesive properties of the haemostaticproduct can be optimized for a certain application by selecting anEL-POX having the optimum density of electrophilic groups. Also otherproperties of the EL-POX, such as hydrophilic/hydrophobic balance andlower critical solution temperature can suitably be optimized bymodifying the concentration and/or properties of the pendant groups.

DETAILED DESCRIPTION THE INVENTION

Accordingly, one aspect of the invention relates to a process ofpreparing a tissue-adhesive haemostatic product, said process comprisingthe steps of:

-   -   providing a porous solid substrate;    -   coating the substrate with a coating liquid that comprises an        electrophilically activated polyoxazoline (EL-POX) and a solvent        to produce a coated substrate, said EL-POX containing at least 2        reactive electrophilic groups;    -   removing the solvent from the coated substrate.

As used herein, the term “tissue-adhesive” refers to the ability of thehaemostatic product to cling to tissue due to the formation of covalentbonds between said product and the tissue. In case of the present tissueadhesive haemostatic product adhesion to tissue may require the presenceof water.

The term “porous” as used herein, unless otherwise indicated, means thatthe haemostatic product comprises pores and/or interstices that admitthe entry of liquid into the product.

The term “porosity” refers to a measure of the void (i.e., “empty”)spaces in the substrate or the haemostatic product, and is percentagevolume of voids over the total volume. The porosity of the haemostaticproducts of the present invention can suitably be determined by methodsknown in the art, such as gas adsorption analysis. Gas adsorptionanalysis involves exposing solid porous materials to gases or vapors ata variety of conditions and evaluating either the weight uptake or thechange in sample volume. Analysis of these data provides informationregarding the physical characteristics of the solid including: skeletaldensity, porosity and total pore volume. Skeletal density is typicallyevaluated by helium pycnometry experiments and represents the true soliddensity of a material when there is no closed porosity. It should beunderstood that in case the substrate is composed of more than one item,porosity refers to the average porosity of the individual items. Thus,if the substrate is a porous powder, porosity equals the percentage ofthe volume of the porous particles that is occupied by pores.

The term “mean pore size” as used herein refers to the mean porediameter as determined by scanning electron microscopy (SEM). A suitablemethod is described in Faraj et al., Tissue Engineering, 2007, 13, 10,2387-2394.

The term “water absorption capacity” as used herein is a measure of thecapability of the porous solid substrate to absorb water. The waterabsorption capacity of the porous solid substrate is determined byweighing a sample of the dry porous substrate (weight=W_(d)) followed byimmersion of the porous substrate into distilled water (37° C.) for 45minutes. Next, the sample is removed from the water and water clingingto the outside of the substrate is removed, following which the sampleis weighed again (weight=W_(w)). The water absorptioncapacity=100%×(W_(w)−W_(d))/W_(d). The water adsorption capacity isindicative of the porosity of the substrate as well as of its ability toswell in the presence of water.

The term “polyoxazoline” as used herein refers to apoly(N-acylalkylenimine) or a poly(aroylalkylenimine) and is furtherreferred to as POX. An example of POX is poly(2-ethyl-2-oxazoline). Theterm “polyoxazoline” also encompasses POX copolymers.

The term “electrophilic group” refers to a functional group that issusceptible to nucleophilic attack from a nucleophilic group and that iscapable of reacting with such a nucleophilic group under the formationof a covalent bond. Electrophilic groups are typically positivelycharged and/or electron/deficient.

The term “nucleophilic group” refers to a functional group that issusceptible to electrophilic attack from an electrophilic group and thatis capable of reacting with an electrophilic group under the formationof a covalent bond. Nucleophilic groups typically are electron rich,having an unshared pair of electrons acting as a reactive site.

The term “activated”, unless otherwise indicated, refers to amodification of a polymer to generate or introduce a new reactivefunctional group wherein the new reactive functional group is capable ofundergoing reaction with another functional group to form a covalentbond.

The term “cross-linked” as used herein refers to components such aspolymers which are intermolecularly bound by covalent bonds. Covalentbonding between two crosslinkable components may be direct, or indirectthrough a linking group.

The term “buffering system” as used herein refers to a substance or acombination of substances that can be used in aqueous systems to drive asolution to a certain buffering pH and wherein the buffering system hasa capacity to prevent change in this buffering pH.

The “buffering pH” of a liquid as used herein refers to the pH value at20° C. measured after the liquid has been diluted 10 times withdistilled water.

The “buffer capacity” as used herein refers to the capability of liquidto resist changes in pH. The buffer capacity β of a liquid (coatingliquid or buffering liquid) is measured at 20° C. after 10 timesdilution with distilled water and expressed in mmol·l⁻¹·pH⁻¹. The buffercapacity is defined as follows:

$\beta = \frac{dn}{d( {p\lbrack H^{+} \rbrack} )}$where dn is an infinitesimal amount of added base and d(p[H⁺]) is theresulting infinitesimal change in the cologarithm of the hydrogen ionconcentration.

The EL-POX employed in accordance with the present invention ispreferably derived from a polyoxazoline whose repeating units arerepresented by the following formula (I):(CHR¹)_(m)NCOR²R², and each of R¹ independently being selected from H, optionallysubstituted C₁₋₂₂ alkyl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted aryl; and m being 2 or 3.

According to a preferred embodiment, the polyoxazoline is a polymer,even more preferably a homopolymer of 2-alkyl-2-oxazoline, said2-alkyl-2-oxazoline being selected from 2-methyl-2-oxazoline,2-ethyl-2-oxazoline, 2-propyl-2-oxazoline, 2-butyl-2-oxazoline andcombinations thereof. Preferably, the polyoxazoline is a homopolymer of2-propyl-2-oxazoline and more preferably of 2-ethyl-oxazoline.

EL-POX can carry electrophilic groups in its side chains (also referredto as pendant electrophilic groups), at its termini, or both. An exampleof a terminal, end capped, EL-POX is a succinimidyl succinate ester likeCH₃O—POX—O₂C—CH₂—C(CH₂CO₂—NHS)₃. An example of a side chain activatedEL-POX is POX containing NHS groups in the alkyl side chain. Yet anotherexample of EL-POX are star-shaped POX-polymers end-functionalized withan NHS-esters.

The electrophilic groups present in the EL-POX are preferably selectedfrom: carboxylic acid esters, sulfonate esters, phosphonate esters,pentafluorophenyl esters, p-nitrophenyl esters, p-nitrothiophenylesters, acid halide groups, anhydrides, ketones, aldehydes, isocyanato,thioisocyanato (isothiocyanato), isocyano, epoxides, activated hydroxylgroups, olefins, glycidyl ethers, carboxyl, succinimidyl esters,succinimidyl carbonates, succinimidyl carbamates, sulfosuccinimidylesters, sulfosuccinimidyl carbonates, maleimido (maleimidyl),ethenesulfonyl, imido esters, aceto acetate, halo acetal, orthopyridyldisulfide, dihydroxy-phenyl derivatives, vinyl, acrylate, acrylamide,iodoacetamide and combinations thereof. More preferably, theelectrophilic groups present in the EL-POX are selected from: carboxylicacid esters, acid chloride groups, anhydrides, ketones, aldehydes,isocyanato, thioisocyanato, epoxides, activated hydroxyl groups,olefins, carboxyl, succinimidyl ester, succinimidyl carbonate,succinimidyl carbamates, sulfosuccinimidyl ester, sulfosuccinimidylcarbonate, maleimido, ethenesulfonyl and combinations thereof. Even morepreferably, the electrophilic groups present in the EL-POX are selectedfrom aldehydes, isocyanato, thioisocyanato, succinimidyl ester,sulfosuccinimidyl ester, maleimido and combinations thereof. Mostpreferably, the electrophilic groups present in the EL-POX are selectedfrom isocyanato, thioisocyanato, succinimidyl ester, sulfosuccinimidylester, maleimido and combinations thereof.

Examples of sulfonate esters that can be used as electrophilic groupsinclude mesylate, tosylate, nosylate, triflate and combinations thereof.Examples of olefins that can be employed include acrylate, methacrylate,ethylacrylate and combinations thereof. Examples of activated hydroxylgroups include hydroxyl groups that have been activated with anactivating agent selected from p-nitrophenyl chlorocarbonates,carbonyldiimidazoles (e.g. 1,1-carbonyl diimidazole) and sulfonylchloride.

The EL-POX employed in the present method preferably contains at least10 reactive electrophilic groups. More preferably, the EL-POX containsat least 25, even more preferably at least 35 and most preferably atleast 50 reactive electrophilic groups.

The EL-POX of the present invention advantageously contains one or morependant electrophilic groups. Typically, the EL-POX contains 3 to 50pendant electrophilic groups per 100 monomers, more preferably 4 to 35pendant electrophilic groups per 100 monomers, even more preferably atleast 5 to 25 pendant electrophilic groups per 100 monomers.

The EL-POX employed in accordance with the present invention typicallyhas an average molecular weight in the range of 1,000 to 100,000 g/mol,more preferably of 5,000 to 50,000 and most preferably of 10,000 to30,000 g/mol.

In one embodiment of the invention the porous solid substrate containsat least 50 wt. %, most preferably at least 80 wt. % of a polysaccharideselected from dextran, alginates, oxidized cellulose, oxidizedregenerated cellulose (ORC), hydroxyethylcellulose,hydroxymethylcellulose, hyaluronic acid; and combinations thereof. In aparticularly preferred embodiment the porous solid substrate contains atleast 50 wt. %, most preferably at least 80 wt. % of ORC.

Cellulose is a homopolysaccharide of glucopyranose polymerized throughβ-glucosidic bonds. Prior to oxidation, the cellulose can remainnon-regenerated with unorganized fibers or can be regenerated to formorganized fibers. When cellulose fibers are treated with dinitrogentetroxide, hydroxyl groups are oxidized into carboxylic acid groupsyielding a polyuronic acid. While polyuronic acid is the main componentof oxidized cellulose, nonoxidized hydroxyl groups remain as a fibrouscomponent.

In a further embodiment of the invention the porous solid substratecomprises an outer surface that comprises a nucleophilic polymercontaining reactive nucleophilic groups. Preferably, the porous solidsubstrate contains at least 5 wt. %, more preferably at least 10 wt. %and more preferably at least 50 wt. % of the nucleophilic polymer. Mostpreferably, the substrate consists of said nucleophilic polymer.

The nucleophilic polymer typically contains at least 2 nucleophilicgroups, more preferably at least 10 nucleophilic groups, most preferablyat least 20 nucleophilic groups.

The nucleophilic groups of the nucleophilic polymer are preferablyselected from amine groups, thiol groups, phosphine groups andcombinations thereof. More preferably, these nucleophilic groups areamine groups. These amine groups are preferably selected from primaryamine groups, secondary amine groups and combinations thereof.

The nucleophilic polymer in the outer surface of the porous solidsubstrate preferably is a nitrogen rich polymer having a nitrogencontent of at least 1 wt. %, more preferably of 5-10 wt. % and mostpreferably of 15-25 wt. %.

The nucleophilic polymer is preferably selected from protein, chitosanand synthetic or carbohydrate polymers containing reactive nucleophilicgroups selected from amine, thiol, phosphine and combinations thereof.More preferably, the nucleophilic polymer is selected from collagen,chitosan and combinations thereof.

The term collagen as used herein refers to all forms of collagenincluding processed derivatives. Preferred collagens do not possestelopeptide regions (“atelopeptide collagen”), are soluble, and may bein fibrillar or non-fibrillar form. The collagen can be selected fromthe group of microfibrillar collagen, synthetic human collagen such asthe type I collagen, type III collagen, or a combination of type Icollagen and type III collagen. Collagen crosslinked using heat,radiation, or chemical agents such as glutaraldehyde may also be used toform particularly rigid crosslinked compositions. Dry, porous freezedried collagen sponges are specifically preferred.

Chitosan is a biodegradable, nontoxic, complex carbohydrate derivativeof chitin (poly-[134]-N-acetyl-D-glucosamine), a naturally occurringsubstance. Chitosan is the deacetylated form of chitin. In general, thegeneric term chitosan is applied when the extent of deacetylation isabove 70% and the generic term chitin is used when the extent ofdeacetylation is insignificant, or below 20%. With less than 100%deacetylation, the chitosan polysaccharide is a linear block copolymercontaining both N-acetyl-D-glucosamine and D-glucosamine monomer units.

According to one preferred embodiment, the nucleophilic groups of thenucleophilic polymer present in the surface of the porous solidsubstrate are amine groups and the electrophilic groups comprised in theEL-POX are selected from carboxylic acid esters, sulfonate esters,phosphonate esters, pentafluorophenyl esters, p-nitrophenyl esters,p-nitrothiophenyl esters, acid halide groups, anhydrides, ketones,aldehydes, isocyanato, thioisocyanato, isocyano, epoxides, activatedhydroxyl groups, glycidyl ethers, carboxyl, succinimidyl esters,succinimidyl carbonates, succinimidyl carbamates, sulfosuccinimidylesters, sulfosuccinimidyl carbonates, imido esters, dihydroxy-phenylderivatives, and combinations thereof.

Examples of succinimidyl derivatives that may be employed includesuccinimidyl glutarate, succinimidyl propionate, succinimidylsuccinamide, succinimidyl carbonate, disuccinimidyl suberate,bis(sulfosuccinimidyl) suberate, dithiobis(succinimidylpropionate),bis(2-succinimidooxycarbonyloxy) ethyl sulfone and3,3′-dithiobis(sulfosuccinimidyl-propionate). Examples ofsulfosuccinimidyl derivatives that can be used includesulfosuccinimidyl(4-iodoacetyl)aminobenzoate, bis(sulfosuccinimidyl)suberate,sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate,dithiobis-sulfosuccinimidyl propionate, disulfo-succinimidyl tartarate;bis[2-(sulfo-succinimidyloxycarbonyloxyethylsulfone)], ethylene glycolbis(sulfosuccinimiclylsuccinate), dithiobis-(succinimidyl propionate).Examples of dihydroxyphenyl derivatives include dihydroxyphenylalanine,3,4-dihydroxyphenylalanine (DOPA), dopamine, 3,4-dihydroxyhydroccinamicacid (DOHA), norepinephrine, epinephrine and catechol.

According to another preferred embodiment, the nucleophilic groups ofnucleophilic polymer in the outer surface of the porous solid substrateare thiol groups and the electrophilic groups contained in the EL-POXare selected from halo acetals, orthopyridyl disulfide, maleimides,vinyl sulfone, dihydroxyphenyl derivatives, vinyl, acrylate, acrylamide,iodoacetamide, succinimidyl ester, succinimidyl carbonate, succinimidylcarbamates, sulfosuccinimidyl ester, sulfosuccinimidyl carbonate andcombinations thereof. More preferably, the electrophilic groups areselected from succinimidyl esters, halo acetals, maleimides, ordihydroxyphenyl derivatives and combinations thereof. Most preferably,electrophilic groups are selected from maleimides or dihydroxyphenylderivatives and combinations thereof.

The porous solid substrate that is employed in the present processpreferably has a porosity of at least 5 vol. %. In case the substrate isa foam or a mesh, the substrate preferably has a porosity of at least 50vol. %, more preferably of at least 70 vol. % and most preferably of atleast 85 vol. %. In case the substrate is a porous powder, porositypreferably is at least 20 vol. %, more preferably at least 50 vol. % andmost preferably at least 75 vol. %.

The porous solid substrate that is employed in the present processpreferably has a mean pore size of at least 2 μm. In case the substrateis a foam or a mesh, the substrate preferably has a mean pore size 5 to500 μm, preferably of 10 to 200 μm. In case the substrate is a porouspowder, the mean pore size is preferably 4 to 50 μm, more preferably 6to 25 μm.

The porous solid substrate typically has a water absorption capacity ofat least 25%, more preferably of at least 100%, even more preferably ofat least 250% and most preferably of at least 1000%.

The porous solid substrate that is employed in the present processpreferably is an object in the form of a mesh or a foam, said objecthaving a shape that facilitates application of the coated substrate as awound dressing, e.g. a sheet. Typically, the substrate has a length of10 mm to 200 mm, a width of 5 mm to 200 mm and a thickness of 0.5 mm to10 mm.

In one embodiment of the present invention the porous solid substrate isa mesh. An example of a mesh is a sheet or gauze made from woven ornon-woven fibres. The fibres contained in the mesh are preferably madeof biocompatible and biodegradable polymers, like gelatin, collagen, ORCor combinations thereof.

In another embodiment of the present invention the porous solidsubstrate is a solid foam, sometimes also referred to as sponges. Thesolid foam is preferably made of cross linked gelatin (gelfoam).

In a further embodiment of the invention the porous solid substrate is apowder. The porous powder is preferably made of gelatin orpolysaccharide. Suitable polysaccharides are starch, modified starches,alginates, chitosan, dextran and combinations thereof. Most preferably,the polysaccharide employed is modified starch.

Preferably the porous powder is in the form of a free flowing, sterilepowder. Advantageously, the powder is a micro porous powder.

The porous powder typically has a mass weighted average particle size inthe range of 10-200 μm, more preferably of 25-100 μm and most preferablyof 50-75 μm.

The coating liquid employed in the present process preferably containsat least 50 wt. %, more preferably at least 60 wt. %, most preferably atleast 80 wt. % of solvent. The solvent is preferably selected from2-propanol, ethyl alcohol, methanol, dichloromethane, acetone, anisole,1-butanol, 2-butanol, butyl acetate, tert-butylmethyl ether, cumene,dimethyl sulfoxide, ethyl acetate, ethyl ether, ethyl formate, heptane,isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol,methylethylketone, methylisobutylketone, 2-methyl-1-propanol, pentane,1-pentanol, 1-propanol, propyl acetate and combinations thereof. Morepreferably the solvent is selected from 2-propanol, ethyl alcohol,methanol, acetone and combinations thereof.

The coating liquid may suitably contain some water. Typically, thecoating liquid contains less than 5 wt. % water, more preferably lessthan 1 wt. % water.

The coating liquid employed in the present process preferably containsat least 1 wt. % of EL-POX. More preferably the EL-POX content of thecoating liquid is at least 5 wt. %, even more preferably at least 10 wt.% and most preferably at least 20 wt. %.

According to a preferred embodiment of the invention the porous solidsubstrate is coated by spraying the coating liquid onto the substrate.In accordance with a particularly preferred embodiment, the coatingliquid is sprayed onto the substrate through an ultrasonic nozzle. Theinventors have found that the use of an ultrasonic nozzle makes itpossible to homogeneously coat the substrate with a coating liquid thatcontains a considerable amount of EL-POX.

Solvent can suitably be removed from the coated substrate byevaporation. Evaporation preferably is conducted under reduced pressure,e.g. at a pressure of less than 1 mbar.

Alternatively, the solvent can be removed from the coated substrate bycontacting the coated substrate with a liquefied gas or a super criticalfluid. Preferably, the liquefied gas or the supercritical fluid has apressure of at least 30 bar.

In accordance with an advantageous embodiment of the present invention,the porous solid substrate comprises an outer surface that comprises anucleophilic polymer as described herein before, and following thecoating of the substrate with the coating liquid and before and/orduring the removal of the solvent, the reactive electrophilic groups ofthe EL-POX react with the reactive nucleophilic groups of thenucleophilic polymer under formation of covalent bonds. Preferably saidreaction occurs at a temperature of less than 50° C., more preferably ata temperature in the range of 15-25° C. (ambient conditions). Thisembodiment of the present process offers the advantage that the reactionbetween the reactive electrophilic groups and the reactive nucleophilicgroups can occur in the absence of a buffering system.

In the present process the reactive electrophilic groups in the EL-POXmay react with reactive nucleophilic groups present in the porous solidsubstrate and/or with nucleophilic groups present in other componentsthat are employed in the process (e.g. nucleophilic crosslinkers).Preferably, after the removal of the solvent from the coated substrate,the EL-POX still contains on average at least one, more preferably atleast 5 and most preferably at least 40 reactive electrophilic groups.These reactive electrophilic groups provide adhesive properties to thecoated substrate as they can form covalent bonds with e.g. amine groupsnaturally present in tissue.

The coating liquid employed in the present process may contain theEL-POX in dissolved and/or dispersed form.

In accordance with one embodiment of the invention the coating liquidcontains EL-POX in fully dissolved form and the coating liquid furthercontains a dispersed buffering system. Preferably, the coating liquidhas a buffering pH in the range of 7 to 11, more preferably in the rangeof 8 to 10. The buffer capacity of the coating liquid preferably is atleast 10 mmol·l⁻¹·pH⁻¹. More preferably, the buffer capacity is a least25 mmol·l⁻¹·pH⁻¹, most preferably the buffer capacity is at least 50mmol·l⁻¹·pH⁻¹.

In another embodiment of the invention the coating liquid containsEL-POX in fully dissolved form and the process comprises covering theporous solid substrate with a buffer liquid before the substrate iscoated with the EL-POX containing coating liquid, said buffer liquidcomprising a buffering system. Preferably, the buffer liquid has abuffering pH in the range of 7 to 11, more preferably in the range of 8to 10. The buffer capacity of the buffer liquid preferably is at least10 mmol·l⁻¹·pH⁻¹. More preferably, the buffer capacity is a least 25mmol·l⁻¹·pH⁻¹, most preferably the buffer capacity is at least 50mmol·l⁻¹·pH⁻¹. In case the buffer liquid is aqueous, the substrate ispreferably dried after the covering with the buffer liquid before thecoating with the coating liquid. Thus, unwanted cross-linking betweenthe EL-POX and the substrate and decomposition of the EL-POX can beminimized. This embodiment offers the advantage that the EL-POX canpenetrate the pores of the porous solid substrate and form a coatinginside these pores. The buffer liquid employed in accordance with thisembodiment may suitably contain a nucleophilic cross-linking agent, saidnucleophilic cross-linking agent containing at least 2 reactivenucleophilic groups.

In yet another embodiment at least 80 wt. % of the EL-POX present in thecoating liquid is undissolved when the coating liquid is coated on theporous solid substrate. Advantageously, besides the undissolved EL-POXthe coating liquid contains a dissolved or undissolved buffering system.The coating liquid preferably has a buffering pH in the range of 7 to11, more preferably in the range of 8 to 10. The buffer capacity of thecoating liquid preferably is at least 10 mmol·l⁻¹·pH⁻¹. More preferably,the buffer capacity is a least 25 mmol·l⁻¹·pH⁻¹, even more preferablythe buffer capacity is at least 50 mmol·l⁻¹·pH⁻¹. Preferably, after thecoating of the substrate with the coating liquid containing undissolvedEL-POX, the substrate is covered with a liquid solvent composition inwhich the EL-POX is soluble. This embodiment offers the advantage thatthe EL-POX does not enter the pores and that the EL-POX coating layer isconcentrated onto the surface of the porous substrate. According to aparticularly preferred embodiment, the liquid solvent compositioncontains a nucleophilic cross-linking agent.

Examples of nucleophilic cross-linking agents that may suitably beemployed include nucleophilically activated PEG, nucleophilicallyactivated POX, trilysine and combinations thereof.

The nucleophilic cross-linking agent preferably contains at least 3reactive nucleophilic groups. The nucleophilic groups of thenucleophilic cross-linking agent are preferably selected from aminegroups, thiol groups, phosphine groups and combinations thereof. Morepreferably, these nucleophilic groups are amine groups. According to apreferred embodiment, the nucleophilic groups present in thenucleophilic cross-linking agent are primary amine groups.

In one embodiment of the invention the nucleophilic cross-linking agentis a low molecular weight polyamine having a molecular weight of lessthan 1,000 g/mol, more preferably of less than 700 g/mol and mostpreferably of less than 400 g/mol. Even more preferably, thenucleophilic cross-linking agent is selected from the group of dilysine;trilysine; tetralysine; pentalysine; dicysteine; tricysteine;tetracysteine; pentacystein; oligopeptides comprising two or more aminoacid residues selected from lysine, omithine, cysteine, arginine andcombinations thereof, and other amino acid residues; spermine;tris(aminomethyl)amine; arginine and combinations thereof.

According to another embodiment of the invention, the nucleophiliccross-linking agent is a high molecular weight polyamine selected fromthe group of: nucleophilically activated POX (NU-POX) comprising atleast two amine groups; chitosan; chitosan derivatives (e.g.dicarboxy-derivatised chitosan polymers as described in WO 2009/028965),polyethyleneimines; polyvinylamine; polyallyl amine;amine-functionalized poly(meth)acrylates; polysaccharides containingamine-functional moieties such as aminoglycosides, such as4,6-disubstituted deoxystreptamine (Kanamycin A, Amikacin, Tobramycin,Dibekacin, Gentamicin, Sisomicin, Netilmicin), 4,5-disubstituteddeoxystreptamine (Neomycins B, C and Neomycin E (paromomycin)) andnon-deoxystreptamine aminoglycosides, e.g. streptomycin; styrenics;polypeptides comprising two or more amino acid residues selected fromlysine, omithine, cysteine, arginine and combinations thereof, and otheramino acid residues; and combinations thereof. Albumin of natural sourceor recombinant is an example of a polypeptide that may suitably beemployed as a polypeptide. Amine-functionalized polyethylene glycol isanother example of a high molecular weight polyamine that can suitablybe employed as the nucleophilic cross-linking agent.

According to another preferred embodiment, the nucleophilic groupspresent in the nucleophilic cross-linking agent are thiol (sulfohydryl)groups.

In one embodiment of the invention the nucleophilic cross-linking agentemployed in the cross-linked polymer is a low molecular weight polythiolcomprising 2 or more thiol groups having a molecular weight of less than1,000 g/mol, more preferably of less than 700 g/mol and most preferablyof less than 400 g/mol. Even more preferably, the nucleophiliccross-linking agent is selected from the group of trimercaptopropane,ethanedithiol, propanedithiol, 2-mercaptoethyl ether,2,2′-(ethylenedioxy)diethanethiol, tetra(ethylene glycol) dithiol,penta(ethylene glycol) dithiol, hexaethylene glycol dithiol; thiolmodified pentaerythritol, dipentaerythritol, trimethylolpropane orditrimethylolpropane; oligopeptides containing at least two cysteineunits.

According to another embodiment of the invention, the nucleophiliccross-linking agent employed in the cross-linked polymer is a highmolecular weight polythiol selected from the group of: NU-POX comprisingat least two thiol groups; thiol-functionalized poly(meth)acrylates;polysaccharides containing thiol-functional moieties; styrenics;polypeptides comprising two or more thiol groups.

The nucleophilic cross-linking agent, as defined herein before, maysuitably be employed in the coating liquid and/or the buffer liquid tocovalently link the EL-POX to a porous solid substrate with an outersurface that comprises a polymer containing reactive electrophilicgroups. Suitable polymers with electrophilic groups arepoly(lactic-co-glycolic acid), chondroitin sulfate-NHS, chondroitinsulfate succinimidyl succinate, alginate-NHS, hyaluronic acid-NHS,copolymers comprising N-hydroxy succinimide carbonate containingmethacrylate monomers (as described in Cengiz et al., 2010, J. ofPolymer Science Part A: Polymer Chemistry, vol. 48, issue 21,4737-4746), biological low-molecular-weight derivatives obtained bymodifying at least one carboxyl group of a biologicallow-molecular-weight, compound having two or more carboxyl groups, withN-hydroxysuccinimide, N-hydroxysulfosuccinimide, or a derivative thereof(as described in EP1548004). Preferably, the nucleophilic cross-linkingagent is employed in the coating liquid.

In another preferred embodiment of the invention, the EL-POX and thenucleophilic cross-linking agent are present in separate fluids, butcoated simultaneously onto the porous solid substrate. This may suitablybe achieved by using two ultrasonic spray nozzles. Accordingly, themethod according to this advantageous embodiment comprises:

-   -   providing a solid porous substrate,    -   providing the coating liquid, comprising EL-POX in a first        solvent;    -   providing a second liquid comprising a buffering system and/or a        nucleophilic cross-linking agent, as well as a second solvent;    -   creating a first spray stream by passing the coating liquid        through a first ultrasonic nozzle;    -   creating a second spray stream by passing the second liquid        through a second ultrasonic nozzle;    -   simultaneously exposing at least a part of the surface of the        solid porous solid substrate to both the first spray stream and        the second spray stream to cover said part of the solid porous        substrate with a coating mixture that comprises the EL-POX as        well as the nucleophilic cross-linking agent and/or the        buffering system;    -   removing the first and second solvent from the coating mixture        to obtain a porous solid substrate that is at least partly        coated with a dry layer containing the EL-POX and the        nucleophilic cross-linking agent.

The EL-POX in this method may suitably be replaced byPVP-acrylic-acid-NHS or (poly-((N-vinylpyrrolidone)₅₀-co-(acrylicacid)₂₅-co-(acrylic acid N-hydroxysuccinimide ester)₂₅).

In case a nucleophilic cross-linking agent is employed in the abovedescribed dual spray method, the EL-POX and the nucleophiliccross-linking agent may react with each other to form a cross-linkedpolymer before, during or after removal of the solvents. In a preferredembodiment, the cross-linked polymer that is comprised in the dry layercomprises unreacted electrophilic groups. Suitable solvents are asdefined herein before. In a preferred embodiment, the first and secondsolvent are miscible, in a more preferred embodiment the first andsecond solvent are the same.

In case the second liquid contains a buffering system, the second liquidpreferably has a buffering pH in the range of 7 to 11, more preferablyin the range of 8 to 10. The buffer capacity of the second liquidpreferably is at least 10 mmol·l⁻¹·pH⁻¹. More preferably, the buffercapacity is a least 25 mmol·l⁻¹·pH⁻¹, most preferably the buffercapacity is at least 50 mmol·l⁻¹·pH⁻¹.

Another aspect of the invention relates to an adhesive haemostaticproduct selected from a coated mesh, a coated foam or a coated powder,said haemostatic product comprising:

-   -   a porous solid substrate having a porosity of at least 20 vol. %        and comprising an outer surface that comprises a nucleophilic        polymer containing reactive nucleophilic groups;    -   an adhesive coating that covers at least a part of the solid        substrate, said coating comprising EL-POX containing on average        at least 1 reactive electrophilic group.

This adhesive haemostatic product may suitably be obtained by theprocess described herein before.

As explained herein before, the haemostatic product has excellentadhesive properties due to the presence of electrophilic reactive groupsthat are capable of reacting with nucleophilic groups that are naturallypresent in tissue. In addition, this haemostratic product offers theadvantage that upon contact with blood or other aqueous fluids, theadhesive coating will be anchored to the porous substrate as reactiveelectrophilic groups in the EL-POX will react with reactive nucleophilicgroups of the nucleophilic polymer under the formation of covalentbonds.

The EL-POX present in the coating of the adhesive haemostatic materialmay be covalently bound to the porous solid substrate. Furthermore, theEL-POX may be cross-linked. Preferred forms of the porous solidsubstrate, the EL-POX and the nucleophilic polymer have been describedherein before.

The EL-POX in the coating of the haemostatic product preferably contains5 reactive electrophilic groups, more preferably 25 reactiveelectrophilic groups and most preferably 50 reactive electrophilicgroups.

The substrate typically represents at least 10 wt. %, more preferably atleast 50 wt. % and most preferably at least 75 wt. % of the haemostaticproduct.

The EL-POX containing coating typically represents 5-75 wt. % of thehaemostatic product. More preferably, the coating represents 10-50 wt.%, most preferably 12-25 wt. % of the haemostatic product.

The coating preferably contains 25-100 wt. % of EL-POX. More preferably,the EL-POX content of the coating is at least 50 wt. %, most preferablyat least 75 wt. %.

The present invention enables the preparation of coated haemostaticproducts wherein the adhesive coating contains pores that areinterconnected with the pores of the porous solid substrate.

According to a particularly preferred embodiment, the adhesivehaemostatic has a pore density of at least 50%, more preferably of atleast 80%

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1

NHS-side chain activated poly[2-(propyl/NHS-ester-ethyl)-2-oxazoline]copolymer, containing 25% NHS ester (=EL-POX, 25% NHS) units wassynthesized as follows: Apoly[2-(propyl/methoxy-carbonyl-ethyl)-2-oxazoline] copolymer (Degree ofpolymerization=DP=around 100) was synthesized by making use of cationicring opening polymerization (CROP) using 75% 2-n-propyl-2-oxazoline(nPropOx) and 25% 2-methoxycarbonyl-ethyl-2-oxazoline (MestOx). Astatistical copolymer containing 25% 2-methoxycarbonyl-ethyl groups(¹H-NMR) was obtained. The polymer containing 25%2-methoxycarbonyl-ethyl groups, was hydrolyzed using sodium hydroxide(1M), yielding a copolymer with 25% 2-carboxy-ethyl-groups (¹H-NMR). The2-carboxy-ethyl-groups were activated by N-hydroxysuccinimide (NHS) anddiisopropylcarbodiimide (DIC), yielding a2-(propyl/NHS-ester-ethyl)-2-oxazoline] copolymer (=EL-POX, 25% NHS).The polymer contained 25% NHS-ester groups according to ¹H-NMR andUV-spectroscopy.

Bovine collagen sponges were prepared according to the proceduredescribed in Faraj et al., Tissue Engineering, 2007, 13, 10, 2387-2394.The collagen was extracted from the tendon of a cow. The collagensponges so obtained had a porosity of 95% to 98% and average pore sizesof 80-100 μm. The porosity was calculated by comparing the density of acollagen film (without void spaces) with the density of the collagensponges. The pore sizes were determined using scanning electronmicroscopy according to the method described in Faraj et al., TissueEngineering, 2007, 13, 10, 2387-2394.

The EL-POX, 25% NHS was dissolved in acetone (180 mg/ml). The solutionwas evenly distributed drop wise on top of the freeze dried bovinecollagen sponges. Immediately after coating, the sponges were dried atroom temperature under vacuum (1 mbar) for 8 hours. Adhesive collagensponges with an EL-POX, 25% NHS coating (15 mg/cm²) were obtained.

To assess possible cross-linking between the EL-POX, 25% NHS and thenucleophilic amine groups present on the collagen, the coated collagensponge was rinsed with acetone. All EL-POX, 25% NHS was recovered in theacetone extract, indicating that the electrophilic groups in the EL-POXhad not reacted with the amine groups in the collagen during coatingand/or drying.

The haemostatic properties of the coated collagen sponges were assessedas follows:

-   -   100 μL of fresh heparinized whole blood was added drop wise on        top of the EL-POX, 25% NHS coated side of a collagen sponge.    -   Another EL-POX, 25% NHS coated sponge was placed on top of the        blood coated collagen sponge, with the EL-POX coated side facing        the blood (‘sandwich method’).    -   Mild pressure was applied for 10 seconds using a gauze. Next,        the sandwich was put in beaker containing water and the water        was stirred for two minutes. The two collagen sponges remained        adhered under these conditions and no blood leaked from the        sponges, indicating haemostasis and adhesion.

In a control experiment using non-coated collagen sponges, the spongesdetached after 20 seconds under water and the water turned red,indicating absence of haemostasis.

Example 2

NHS-side chain activated poly2-(propyl/hydroxy-ethyl-amide-ethyl/NHS-ester-ethyl-ester-ethyl-amide-ethyl)-2-oxazoline]terpolymer containing 15% NHS-ester groups (=EL-POX, 15% NHS) wassynthesized as follows:

Poly[2-(propyl/methoxy-carbonyl-ethyl)-2-oxazoline] copolymer(DP=+/−100) was synthesized by means of CROP using 70%2-propyl-2-oxazoline and 30% 2-metoxycarbonyl-ethyl-2-oxazoline. Astatistical copolymer containing 30% 2-methoxycarbonyl-ethyl groups(¹H-NMR) was obtained. Secondly, the polymer containing 30%2-methoxycarbonyl-ethyl groups, was reacted with ethanolamine yielding acopolymer with 30% 2-hydroxy-ethyl-amide-ethyl-groups (¹H-NMR). Afterthat, part of the 2-hydroxy-ethyl-amide-ethyl-groups was reacted withsuccinic anhydride yielding a terpolymer with 70% 2-propyl groups, 15%2-hydroxy-ethyl-amide-ethyl-groups and 15%2-carboxy-ethyl-ester-ethyl-amide-ethyl-groups according to ¹H-NMR.Lastly, the 2-carboxy-ethyl-ester-ethyl-amide-ethyl-groups wereactivated by N-hydroxysuccinimide (NHS) and diisopropylcarbodiimide(DIC), yielding EL-POX, 15% NHS. The polymer contained 15% NHS-estergroups according to ¹H-NMR.

The EL-POX, 15% NHS (130 mg) and anhydrous sodium borate (47 mg) wereweighed and a solution of isopropanol/2-butanone (1.6 mL, v/v, 1:1) wasadded, resulting in a fine suspension as the anhydrous sodium borate isinsoluble in isopropanol/2-butanone (v/v, 1:1). The suspension wasevenly distributed drop wise on top of the freeze dried bovine collagensponges. Immediately after coating, the sponges were dried at roomtemperature under vacuum (1 mbar) for 8 hours. Adhesive collagen spongeswith an EL-POX, 15% NHS and an anhydrous sodium borate coating (19mg/cm²) were obtained.

The haemostatic properties of the coated collagen sponges of wereassessed as follows:

-   -   100 μL of fresh heparinized whole blood was added drop wise on        top of the EL-POX, 15% NHS and an anhydrous sodium borate coated        side of a collagen sponge.    -   Another EL-POX, 15% NHS and an anhydrous sodium borate coated        sponge was placed on top of the blood coated collagen sponge,        with the EL-POX coated side facing the blood (‘sandwich        method’).    -   Mild pressure was applied for 10 seconds using a gauze. Next,        the sandwich was put in beaker containing water and the water        was stirred for three minutes.

The two collagen sponges remained adhered under these conditions and noblood leaked from the sponges, indicating haemostasis and adhesion.

Example 3

In this example several polymers were tested for shear strength, anoverview of the samples is provided in Table 1. Polymers (DP=+/−100)were synthesized with CROP of 2-n-propyl-2-oxazoline,2-ethyl-2-oxazoline and 2-methoxy-ethyl-2-oxazoline. The EL-POX used assample 1-3, were post modified via the route described in example 1. TheEL-POX used as sample 4-6 were synthesized via the route described inexample 2. All activated polymers were analyzed via ¹H-NMR andUV-spectroscopy for NHS content. PEG 4-arm NHS (Pentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate, sample 7) wasobtained from NOF America corporation. Bovine collagen sponges (5×7×1 cm(b×l×h)) were tested as a control (sample 8). All coated sponges wereprepared as described in Example 1.

Preparation Procedure

-   -   Porous collagen sponges were weighed (n=7).    -   EL-POX solutions were prepared by dissolving a defined amount of        polymer in organic solvent (dichloromethane        (DCM)/isopropylalcohol (IPA) (v/v, 1:1)) to a final        concentration of 300 mg/mL.    -   The EL-POX solution was coated dropwise on a part of the porous        collagen sponges (14 cm²) to obtain the aimed coating density of        15 mg/cm². The coated porous collagen sponges were dried        overnight in a vacuum oven (5 mbar) at room temperature.    -   The coated porous collagen sponges were weighed again and the        coating density was determined. An overview of the used polymers        and coating densities are provided in Table 1.        Test Procedure        These polymers were tested for shear strength in the following        way:    -   The constructs were cut into equal pieces of 5×1 cm (b×l).    -   The coated sides were put together with the adhesive sides        facing each other using 200 μL of heparinized human blood. A        weight (10 g) was applied for 10 seconds for a standardized        pressure.    -   The constructs were allowed to crosslink for defined times: 1        minute (t1) or 15 minutes (t15).    -   At the defined time points the constructs were positioned in a        shear tester (Zwicky Roell, 20 N load cell) and shear strength        was measured until failure.    -   Output: The measured force (N) is divided by the overlapping        area of the constructs (cm²) resulting in the shear strength (in        kPa). The results are listed in Table 1.

TABLE 1 Results of shear strength test Coating density (mg/cm2) Shearstrength (kPa) n = 7 n = 6 t1 and t15 Sample EL-POX Average t1 t15 1P(PropOx-EtOx-NHS) (50-40-10) 14.5 ± 0.8 2.10 ± 1.64 5.29 ± 1.44 2P(PropOx-EtOx-NHS) (40-35-25) 14.0 ± 0.9 3.20 ± 1.53 8.29 ± 1.11 3P(PropOx-EtOx-NHS) (40-50-10) 14.5 ± 0.4 1.91 ± 0.85 4.62 ± 0.60 4P(PropOx-OH-NHS) (70-20-10) 14.2 ± 2.9 3.45 ± 0.43 5.27 ± 1.26 5P(PropOx-OH-NHS) (70-5-25) 15.1 ± 1.3 2.98 ± 0.43 8.20 ± 2.16 6P(PropOx-OH-NHS) (50-40-10) 14.6 ± 0.8 1.29 ± 0.60 4.79 ± 1.74Comparative examples 7 PEG 4-arm NHS 13.0 ± 1.5 3.18 ± 0.32 6.78 ± 1.428 Collagen without coating — 0.79 ± 0.48 1.47 ± 0.46From these results can be concluded that:

-   -   the EL-POX polymers (1-6) show higher shear strength with        increasing crosslinking times.    -   EL-POX polymers (2 and 5) show higher shear strength values than        polymer PEG 4-arm NHS (7) at t15.    -   Control sample (8) shows limited to no shear strength,        indicating that the crosslinking is caused by the NHS-ester        groups.        This example illustrates that EL-POX coated on collagen is        capable to crosslink in the presence of blood creating higher        shear strength than the same concentration of PEG 4-arm NHS        coated on collagen.

Example 4

NHS-side chain activated poly2-(propyl/hydroxy-ethyl-amide-ethyl/NHS-ester-ethyl-ester-ethyl-amide-ethyl)-2-oxazoline]terpolymer containing 20% NHS-ester groups (=EL-POX, 20% NHS) wassynthesized as follows:

Poly[2-(propyl/methoxy-carbonyl-ethyl)-2-oxazoline] copolymer(DP=+/−100) was synthesized by means of CROP using 70%2-propyl-2-oxazoline and 30% 2-methoxycarbonyl-ethyl-2-oxazoline. Astatistical copolymer containing 30% 2-methoxycarbonyl-ethyl groups(¹H-NMR) was obtained. Secondly, the polymer containing 30%2-methoxycarbonyl-ethyl groups, was reacted with ethanolamine yielding acopolymer with 30% 2-hydroxy-ethyl-amide-ethyl-groups (¹H-NMR). Next,part of the 2-hydroxy-ethyl-amide-ethyl-groups were reacted withsuccinic anhydride yielding a terpolymer with 70% 2-propyl groups, 10%2-hydroxy-ethyl-amide-ethyl-groups and 20%2-carboxy-ethyl-ester-ethyl-amide-ethyl-groups according to ¹H-NMR.Lastly, the 2-carboxy-ethyl-ester-ethyl-amide-ethyl-groups wereactivated by N-hydroxysuccinimide (NHS) and diisopropylcarbodiimide(DIC), yielding EL-POX, 20% NHS. The polymer contained 20% NHS-estergroups according to ¹H-NMR.

Amine functionalized NU-POX containing propyl and amine groups in thealkyl side chain were synthesized by CROP of nPropOx and MestOx andsubsequent amidation of the methyl ester side chains with ethylenediamine to yield a poly(2-propyl/aminoethylamidoethyl-2-oxazoline)copolymer (NU-POX). The polymer contained 20% NH₂ according to ¹H-NMR.

Bovine collagen sponges (7×5×1 cm) were used, which were prepared asdescribed in Example 1. For these experiments, an ExactaCoat SCultrasonic spraying device (Sono-Tek) equipped with a heating plate wasused to coat the collagen sponges.

Test 4A: EL-POX in Solution

A solution of EL-POX (EL-POX, 20% NHS) in IPA/2-butanone (v/v, 1:1) (90mg/mL) was homogeneously distributed onto the collagen by ultrasonicspraying, according to the settings shown in Table 2, leading to acoating density of 5 mg/cm². SEM-images showed that the porous structureof the collagen was maintained.

The constructs were cut in pieces (2 cm²) and tested using the shearstrength test at t15 as described in example 3. The results were10.9+/−3.3 kPa (n=4), indicating good adhesion and hemostasis.

TABLE 2 Parameters ultrasonic spraying Nozzle 48 Accumist SubstrateDescription Collagen Bovine Type 1 Coating Area (cm) 5 × 7 cm NozzlePower/Idle Power 6 in test 4A | 3 in test 4B-4D | Dispense rate (ml/min) 1 ml/min Pressure (mbar) 40 mbar Trans Speed (mm/sec) 20 mm/s forexample 5A, 5B, 5C (NU-POX coating) | 40 mm/s for example 5C (EL-POXcoating) and 5D | Height from top of substrate 30 mm (mm) Spacing (mm) 5 mm Coating cycles 2 coating cycles for test 4A | 3 coating cycles fortest 4B | 1 coating cycles for tests 4C and 4D |Test 4B: EL-POX with Suspended Buffer

A solution of EL-POX in IPA/2-butanone (v/v, 1:1) (10 mg/mL) withsuspended HEPES-buffer (1.7 g) was homogeneously distributed onto thecollagen by ultrasonic spraying, according to the settings shown intable 2, leading to a coating density of 5 mg/cm². SEM-images showedthat the porous structure of the collagen was maintained. The constructswere cut in pieces (2 cm²) and tested using the shear strength test att15 as described in example 3. The average shear strength measured was2.6+/−0.8 kPa (n=3), indicating good adhesion and hemostasis.

Test 4C: (1) Solution of NU-POX and Buffer+(2) Solution of EL-POX

A solution of NU-POX in water (10 mg/mL) with HEPES (1.7 g) washomogeneously distributed onto the collagen by ultrasonic spraying,according to the settings shown in table 2. The solvent was allowed toevaporate for 30 min. After this, a solution of EL-POX in IPA/2-butanone(v/v, 1:1) (15 mg/mL) was homogeneously distributed by ultrasoundspraying, according to the settings displayed in table 2, on top of thecoated collagen. After coating, a coating density of 5 mg/cm² (+40 wt. %NU-POX and +60 wt. % EL-POX) was obtained. SEM-images showed that theporous structure of the collagen was maintained. The constructs were cutin pieces (2 cm²) and tested using the shear strength test at t15 asdescribed in example 3. The average shear strength measured was3.9+/−2.9 kPa (n=2), indicating good adhesion and hemostasis.

Test 4D: Solution of Buffer (Suspension of EL-POX)

A suspension EL-POX in IPA/diethylether/triethylamine (v/v/v, 50:50:1)was homogeneously distributed onto the collagen by ultrasonic sprayingaccording to the settings shown in table 2. After coating a coatingdensity of 4 mg/cm² was obtained. SEM-images showed that the porousstructure of the collagen was maintained. The constructs were cut inpieces (2 cm²) and tested using the shear strength test as described inexample 3. The average shear strength measured was 4.2+/−3.5 kPa (n=3),indicating good adhesion and hemostasis.

In addition, the haemostatic properties of the ultrasonically coatedcollagen sponges 4A-4D were assessed as follows:

-   -   100 μL of fresh heparinized whole blood was added drop wise on        top of the EL-POX coated side of a collagen sponge.    -   Another EL-POX coated sponge was placed on top of the blood        coated collagen sponge, with the EL-POX coated side facing the        blood (‘sandwich method’).    -   Mild pressure was applied for 10 seconds using a gauze. Next,        the sandwich was put in beaker containing water and the water        was stirred for two minutes.

The two collagen sponges 4A-4D all remained adhered under theseconditions and no blood leaked from the sponges, indicating haemostasisand adhesion.

Example 5

NHS-side chain activated poly2-(propyl/hydroxy-ethyl-amide-ethyl/NHS-ester-ethyl-ester-ethyl-amide-ethyl)-2-oxazoline]terpolymer (=EL-POX, 20% NHS) was synthesized as described in example 2.

The EL-POX, 20% NHS was dissolved in methanol (180 mg/ml). The solutionwas evenly distributed drop wise on top of an Oxidized RegeneratedCellulose patch (ORC, Gelita-Cel). Immediately after coating, thepatches were dried at room temperature under air flow for 4 hours. ORCwith an EL-POX, 20% NHS coating (12 mg/cm²) were obtained.

The haemostatic properties of the coated ORC were assessed as follows:

-   -   100 μL of fresh heparinized whole blood was added drop wise on        top of the EL-POX, 20% NHS coated side of ORC (1×1 cm).    -   Another EL-POX, 20% NHS coated ORC was placed on top of the        blood coated ORC, with the EL-POX coated side facing the blood        (‘sandwich method’).    -   Mild pressure was applied for 10 seconds using a gauze. Next,        the sandwich was put in beaker containing water and the water        was stirred for five minutes. The two ORC pieces remained        adhered under these conditions for five minutes and the medium        did not turn red.

In a control experiment using non-coated ORC, ORC detached within aminute under water during stirring and the water turned red, indicatingabsence of hemostasis.

Example 6

NHS-side chain activated poly2-(propyl/hydroxy-ethyl-amide-ethyl/NHS-ester-ethyl-ester-ethyl-amide-ethyl)-2-oxazoline]terpolymer (=EL-POX, 20% NHS) was synthesized as described in example 2.Chitosan powder was obtained from Sigma-Aldrich. (degree ofdeacetylation 75-85%, Mn 10,000). Starch powder (HaemoCer®) was obtainedfrom BioCer, Germany.

Powders were coated separately with EL-POX, 20% NHS (EL-POX) in thefollowing way.

-   -   Powder (chitosan or starch) was weighed and coated with a        solution of EL-POX (15 mg/mL) in DCM.    -   The suspension was dried under reduced pressure.    -   After this, the dried coated powder was grinded forming a        homogenously fine powder. The coated chitosan powder contains        about 25 wt. % EL-POX. The coated starch powder contains about        10 wt. % EL-POX.

As controls, grinded uncoated chitosan powder, grinded uncoated starchpowder and grinded EL-POX were tested.

The EL-POX coated powders and the control samples were mixed with (1)carbonate buffer (0.1 M, pH 9) or (2) heparinized human blood (pH 7.4)to assess the heamostatic properties. This was tested in the followingway: the powders were weighed in an Eppendorf tube and mixed with 250 μLof buffer (1) or blood (2). Gelation time was determined by invertingthe tube up and down until a gel was formed.

The amount of materials used and the results of the crosslink tests aregiven in Table 3.

TABLE 3 Results of crosslinking tests (2) Chitosan* Starch* EL-POX* (1)Buffer blood sample (mg) (mg) (mg) pH 9 (μL) (μL) gel time 1 16.5 5.7250  2 min 2 17.2 5.8 250  1 min 3 19.8 250 No gel 4 18.0 250 No gel 520.1 250 45 sec 6 24 250 No gel 7 18.1 2.0 250 No gel 8 20 250 No gel 921 250 No gel 10 17.8 2.0 250  2 min *calculated from the wt % EL-POX inthe coated powders

These results show that chitosan is able to crosslink with EL-POX(sample 2) and that starch is not able to crosslink with EL-POX (sample7). Both, chitosan and starch coated with EL-POX were capable ofcrosslinking with blood (samples 1 and 10) while the non-coated chitosanand starch powders did not form a gel in the presence of buffer and/orblood (samples 3, 4, 8 and 9). EL-POX powder was capable of forming agel by reacting with amines in blood (sample 5), whereas nogel-formation was observed when the EL-POX powder was combined withbuffer (sample 6).

Gels containing EL-POX were found to be stable under water. In contrastthereto blood gels prepared by adding 200 mg starch or chitosan to 250μL blood, were not stable under water.

Example 7

NHS-side chain activated poly2-(propyl/hydroxy-ethyl-amide-ethyl/NHS-ester-ethyl-ester-ethyl-amide-ethyl)-2-oxazoline]terpolymer (=EL-POX, 20% NHS) was synthesized as described in example 3.PEG 4-arm NHS (Pentaerythritol poly(ethyleneglycol)ethertetrasuccinimidyl glutarate, EL-PEG) was obtained from NOF Americacorporation. Bovine collagen sponges (7×5×1 cm) were prepared asdescribed in Example 1.

Two application methods were compared: (I) melt method and (II) coatingliquid method.

(I) Melt Method (Comparative Examples)

A known amount of polymer powders, EL-POX, 20% NHS and EL-PEG, werehomogenously distributed on top of the collagen sponges to obtain thecoating densities indicated in table 4. EL-POX, 20% NHS and EL-PEG wereheated at the temperatures indicated in Table 4, in a preheated oven for5 min to melt the polymer powder. Within this time frame and temperaturesetting, both EL-POX and EL-PEG remained stable.

SEM-images of the prepared constructs showed that the formed polymerfilm sealed off the porous structure of the collagen top layer.

TABLE 4 Settings (I) melt method Polymer Temperature oven (° C.) Coatingdensity (mg/cm²) EL-POX, 20% NHS 140 12 EL-PEG 60 11

(II) Coating Liquid Method

EL-POX, 20% NHS powder was dissolved in IPA/DCM (v/v, 1:1) (15 mg/mL)and the solution was distributed evenly on top of the collagen spongesby drip coating, to obtain a coating density of 12 mg/cm². The spongeswere dried in a vacuum oven for 2 hours. SEM-images showed that theporous structure of the collagen was maintained.

The haemostatic properties of the coated collagen sponges were assessedas follows:

-   -   100 μL of fresh heparinized whole blood was added drop wise on        top of the EL-POX, 20% NHS or EL-PEG coated side of a collagen        sponge.    -   Another coated sponge was placed on top of the blood coated        collagen sponge, with the coated sides facing the blood        (‘sandwich method’).    -   Mild pressure was applied for 10 seconds using a gauze. Next,        the sandwich was put in beaker containing water and the water        was stirred for one minute.        The collagen sponges prepared by melting (both EL-POX and        EL-PEG), detached within a minute indicating absence of        haemostasis and adhesion. The collagen sponges (with EL-POX)        prepared by drip coating remained adhered during 1 minute        indicating haemostasis and adhesion.

This example shows that the application method affects the accessibilityof the pores in the collagen and explains the difference in haemostasisand adhesion between the sponges obtained by melting and the spongesobtained by coating with liquid.

Example 8

Treatment of traumatic liver and spleen rupture is a major challenge fora surgeon. Since the spleen has an excellent blood supply and rupture ofthe spleen is often associated with massive abdominal hemorrhage.

Standardized combined penetrating spleen rupture was inflicted in n=1anesthetized swine (Domestic Pig, Male, Body Weight Range: 40 kg,Adult). A midline laparotomy was performed to access the spleen. Using ascalpel, n=3 (S1 . . . S3) subcapsular standardized lesions (10 mm×10mm) were made.

Three types of haemostatic products were tested, an overview is providedin Table 5.

TABLE 5 Description of tested products Sample Product Description ofProduct S1 EL-POX Collagen sponge coated with EL-POX, 25% NHS (5mg/cm²), prepared as in Example 4 Comparative examples S2 No coatingBovine collagen sponge, prepared as in Example 1 S3 Reference 2 × 2 cmHemopatch ™ (obtained from Baxter Healthcare)

The heamostatic products were applied with gentle pressure. Afterapplication of the product the time to haemostasis was assessed, seeTable 6.

TABLE 6 Results animal model Sample Product applied Result S1 EL-POXTTH: 1 minute, n = 1 Comparative examples S2 No coating TTH: 3 to 4minutes, n = 2 S3 Reference TTH: 1 minute, n = 1 TTH = time tohaemostasis; n = no. of products needed to reach hemostasis

This example shows the haemostatic efficacy of the EL-POX coated spongeon the spleen of a pig. Based on the end points in Table 6, the EL-POXcoated sponge performed equally well in this model in obtaininghemostasis as the reference product (Hemopatch™) and better than thecontrol. Furthermore, it was observed that the blood absorption capacityof the EL-POX coated sponge was superior to that of the referenceproduct. Thus, hemostasis achieved with the EL-POX coated sponge wasaided by a rapid onset of blood coagulation.

The invention claimed is:
 1. An adhesive haemostatic product selectedfrom a coated mesh, a coated foam or a coated powder, the haemostaticproduct comprising: a) porous solid substrate having a porosity of atleast 5 vol. % and comprising an outer surface that comprises anucleophilic polymer comprising reactive nucleophilic groups; b) anadhesive coating that covers at least a part of the solid substrate, thecoating comprising an electrophilically activated polyoxazoline(EL-PDX), the polyoxazoline being a polymer of 2-alkyl-2-oxazolineselected from 2-methyl-2-oxazoline, 2-ethyl-2-oxazoline,2-propyl-2-oxazoline, 2-butyl-2-oxazoline and combinations thereof, andthe polyoxazoline being electrophilically activated by the presence ofat least 5 reactive electrophilic groups, wherein, in the presence ofwater, the electrophilic groups of the EL-PDX react with amine groupsnaturally present in tissue, to form covalent bonds and wherein, uponcontact with blood, the electrophilic groups of the EL-PDX react withthe reactive nucleophilic groups of the nucleophilic polymer to formcovalent bonds.
 2. The adhesive haemostatic product according to claim1, wherein the porous solid substrate has a porosity of at least 50 vol.%.
 3. The adhesive haemostatic product according to claim 1, wherein theEL-PDX contains on average at least 10 reactive electrophilic groups. 4.The adhesive haemostatic product according to claim 1, wherein thereactive electrophilic groups are selected from carboxylic acid esters,sulfonate esters, phosphonate esters, pentafluorophenyl esters,p-nitrophenyl esters, p-nitrothiophenyl esters, acid halide groups,anhydrides, ketones, aldehydes, isocyanato, thioisocyanato, isocyano,epoxides, activated hydroxyl groups, glycidyl ethers, carboxyl,succinimidyl ester, succinimidyl carbonate, succinimidyl carbamates,sulfosuccinimidyl ester, sulfosuccinimidyl carbonate, imido esters,dihydroxyphenylalanine, 3,4-dihydroxyphenylalanine (DOPA), dopamine,3,4-dihydroxyhydroccinamic acid (DOHA), norepinephrine, epinephrine,catechol and combinations thereof.
 5. The adhesive haemostatic productaccording to claim 4, wherein the reactive electrophilic groups areselected from isocyanato, thioisocyanato, succinimidyl ester,sulfosuccinimidyl ester, and combinations thereof.
 6. The adhesivehaemostatic product according to claim 1, wherein the EL-PDX isnon-crosslinked.
 7. The adhesive haemostatic product according to claim1, wherein the porous solid substrate represents at least 50 wt. % ofthe haemostatic product.
 8. The adhesive haemostatic product accordingto claim 1, wherein the EL-PDX containing coating represents 5-50 wt. %of the haemostatic product.
 9. The adhesive haemostatic productaccording to claim 1, wherein the adhesive coating contains 25-100 wt. %of EL-PDX.
 10. The adhesive haemostatic product according to claim 1,wherein at least a part of the EL-PDX is covalently bound to the poroussolid substrate.
 11. The adhesive haemostatic product according to claim1, wherein the EL-PDX has an average molecular weight in the range of1,000 to 100,000 g/mol and wherein the EL-PDX contains 3 to 50 pendantelectrophilic groups per 100 monomers.
 12. The adhesive haemostaticproduct according to claim 1, wherein the nucleophilic polymer isselected from the group consisting of protein, chitosan, syntheticpolymers, and carbohydrate polymers; and in which the reactivenucleophilic groups are selected from the group consisting of amine,thiol, phosphine and combinations thereof.
 13. The adhesive haemostaticproduct according to claim 1, wherein the porous solid substrate is apowder having a mass weighted average particle size in the range of10-200 μm.
 14. The adhesive haemostatic product according to claim 13,wherein the powder has a mass weighted average particle size in therange of 25-100 μm.
 15. The adhesive haemostatic product according toclaim 13, wherein the powder comprises gelatin and/or polysaccharide.16. The adhesive haemostatic product according to claim 15, wherein thepowder comprises gelatin.
 17. The adhesive haemostatic product accordingto claim 1, wherein, wherein the porous solid substrate is a mesh or afoam in the form of a sheet with a length of 10 to 200 mm, a width of 5to 200 mm and a thickness of 0.5 to 10 mm.
 18. The adhesive haemostaticproduct according to claim 17, wherein the porous solid substrate is amesh.
 19. The adhesive haemostatic product according to claim 18,wherein the mesh is a sheet or a gauze made from woven or non-wovenfibres.
 20. The adhesive haemostatic product according to claim 19,wherein the fibres contained in the mesh are made of gelatin, collagen,oxidized regenerated cellulose (ORC) or a combination thereof.